The development of transgenic mouse models for cystic fibrosis (CF) has been an important contribution to our understanding of CF and of CF transmembrane conductance regulator (CFTR) function. However, because of the dissimilarity between mouse and human anatomy and physiology, there are limitations in what can be discerned about CF pathology and in development of CF therapies. In this context, the advent of successful mammalian animal cloning by nuclear transfer has opened the door to a host of possibilities to develop large animal models for inherited diseases like CF. Recent studies investigating oligonucleotide based genomic gene targeting suggest that some of these approaches might be effective for modifying somatic nuclei that act as the source of genetic material for nuclear transplantation. Thus it now appears possible to produce a more appropriate animal model of CF. A candidate as an alternative to the mouse model is the rabbit which is phylogenetically and anatomically more closely related to human than is the mouse. The recent sequencing of major portions of rabbit CFTR (rCFTR) gene has now provided the genetic information necessary to manipulate the rCFTR sequence and generate a transgenic CF animal. The small fragment homologous replacement (SFHR) gene targeting strategy is an oligonucleotide-based approach that has been previously used to modify both human and mouse CFTR by introducing the bp deletion that gives rise to the ?F508, CFTR mutation that predominates in the CF patient population. It is, therefore, well suited to generate transgenic cell lines that can be used for nuclear transplantation. This proposal will employ SFHR to introduce a ?F508 mutation into the rCFTR of rabbit fetal fibroblasts. These transgenic donor cells will be clonally enriched by Fluorescence Activated Cell Sorting (FACS) and then be fused with enucleated rabbit oocytes that will ultimately be used for the generation of a CF rabbit. The transgenic donor cells will be initially screened by PCR and then evaluated by Southern blot hybridization. Transplanted oocytes will also be PCR screened and then tested in vitro for embryogenic potential. Those oocytes that have demonstrated embryogenic potential will be introduced into surrogate mothers. The development of a transgenic CF rabbit will greatly enhance our ability to evaluate CF pathology and will facilitate development of new therapeutic regimens to improve the quality of life of CF patients.